Tangential flow filtration devices and methods for leukocyte enrichment

ABSTRACT

The present invention provides tangential flow filtration devices and methods for enriching a heterogeneous mixture of blood constituents for leukocytes by removal of non-leukocyte blood constituents. In one particular embodiment the device can provide a composition enriched in monocytes. One embodiment includes a remover unit ( 1 ) having a crossflow chamber ( 3 ) separated by a microporous filter ( 5 ) from a filtrate chamber ( 4 ), the remover unit ( 1 ) also having a tangential flow inlet ( 6 ), a fluid outlet ( 7 ) for a fluid enriched in leukocytes and a filtrate outlet ( 8 ).

RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationSer. No. 60/390,730, filed Jun. 19, 2002, which is incorporated hereinby reference in its entirety.

BACKGROUND OF THE INVENTION

Blood cell populations enriched for leukocytes are often desired for usein research or therapy. Typical sources of leukocytes include wholeperipheral blood, leukopheresis or apheresis product, or other lesscommon sources, such as umbilical cord blood. Enrichment of leukocytescan be done in several ways. Typical methods include density stepgradients (e.g., FICOLL-HYPAQUE®, colloidal silica, and the like),elutriation, centrifugation, lysis of erythrocytes by hypotonic shock,and various combinations of these methods. There are disadvantages toeach of these methods, one of which is the need for laborious washingsteps after the enrichment step is performed.

Following enrichment, the cells are typically washed by a repetitiveprocess. The steps generally include placing the enriched cellsuspension into a centrifuge tube and pelleting the cells to the bottomof the tube by use of a centrifuge. The tube is removed from thecentrifuge, and the supernatant is decanted from the pelleted cells. Awash liquid is added to the tube, and the cell pellet is resuspended.These steps are typically repeated 2 to 4 times.

One disadvantage of this washing process is that sequential resuspensionand centrifugation can decrease cell viability and increase cell lysis.Another disadvantage of washing by centrifugation is the opportunity forbacteria or other infectious agents to contaminate the cells. Even ifall the materials are kept sterile, the repeated opening of thecentrifuge tubes, and the exposure of pipettes and bottles of washsolution to the air can result in contamination. The risk ofcontamination is significant enough for some medical regulatory agenciesto demand that only “closed” systems are used for cell handling.

Filtration methods have also been used to remove leukocytes from bloodwhile retaining other blood constituents for later use. Such methodsgenerally trap leukocytes on a filter in a non-recoverable form, whileallowing other blood constituents to pass through the filter and into acollection vessel. For example, filters are available to removeleukocytes from blood so that the incidence of alloimmune reactions isminimized following blood transfusions. This removal is typically doneusing filters which are made of matted plastic fiber mesh. The mesh isusually arranged to trap the leukocytes in a reticulated matrix havingenough depth so that the cells are trapped throughout the depth of thefilter, thereby keeping the filter from clogging, as would occur if theleukocytes were trapped on a planar surface.

In addition to the physical trapping of the cells, the materials andlarge surface area of the filter allow leukocytes to adhere irreversiblyto the surface. Many of these adherent cells are the very ones desiredfor some medical procedures. The resulting combination of trapping andadherence to the filter creates a highly efficient means of removing theleukocytes for disposal prior to blood infusion therapy. However, whenleukocytes are the desired cells, this method of filtration is notadvantageous.

A method that has been useful in the fractionation of various particlesis tangential flow filtration (TFF) or “cross-flow” filtration. TFFrelies on the movement of a fluid parallel to the surface of a porousmembrane filter. The pores of the membrane allow passage of the fluidand of particles within the fluid that are smaller than the pores. Inaddition, the cross-flow (or “tangential” flow) of fluid parallel to thefilter prevents a build-up of particles larger than the pores on thefilter surface.

TFF has been used for the gross separation of various materials. The useof tangential flow filtration in the pharmaceutical field has beenreviewed by Genovesi (J. Parenter. Aci. Technol., 37:81, 1983),including the filtration of sterile water for injection, clarificationof a solvent system, and filtration of enzymes from broths and bacterialcultures. Marinaccio et al. (WO 85/03011) report a process for use inthe removal of particulate blood components from blood forplasmapheresis, and Robinson et al. (U.S. Pat. No. 5,423,738) describethe use of TFF for the removal of plasma from blood, allowing thereinfusion of blood cells and platelets into patients.

In another use, TFF has been reported for the filtration of beer (EP 0208 450), specifically for the removal of particulates such as yeastcells and other suspended solids. Kothe et al. (U.S. Pat. No. 4,644,056)disclose the use of TFF in the purification of immunoglobulins from milkor colostrum, and Castino (U.S. Pat. No. 4,420,398) describes its use inthe separation of antiviral substances, such as interferons, from brothscontaining these substances as well as viral particles and cells.Similarly, TFF has been used in the separation of bacterial enzymes fromcell debris. (Quirk et al., Enzyme Microb. Technol., 6:201, 1984.) Inaddition, tangential flow filtration units have been employed in theconcentration of cells suspended in culture media. (See, e.g., Radlett,J. Appl. Chem. Biotechnol., 22:495, 1972.)

TFF has also recently been reported to separate liposomes and lipidparticles according to size. (Lenk et al., U.S. Pat. No. 5,948,441.) TFFallows for the formation and isolation of liposomes and lipid particleshaving a defined size range from heterogeneous populations of suchparticles. (See Lenk et al., supra).

However, while TFF has been used for gross fractionation of biologicalliquids and the separation of, for example, liposomes, the use of TFFfor separation of different live cell populations having definedcharacteristics has not been appreciated in the art. In particular, theunique problems associated with the selective separation of leukocytepopulations (such as, e.g., monocytes, CD34⁺ hematopoietic stem andprecursor cells, dendritic precursor cells, and the like) from otherblood cells while maintaining sterility, cell viability, potentialhematopoietic to differentiate, and immunotherapeutic cellular activityhas not been addressed. In addition, the removal of other cellpopulations such as, e.g., populations with overlapping size ranges, hasnot been solved by current approaches.

Therefore, there remains a need in the art for additional devices andmethods for selectively enriching leukocytes from other bloodconstituents, including plasma, erythrocytes, and/or platelets, whilepreserving sterility, cell viability, potential to differentiate, andimmunotherapeutic cellular activity. The present invention satisfiesthese and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to the separation of leukocytes from bloodand blood preparations. In particular, a cell population enriched inleukocytes is prepared by the use of a tangential flow filtrationdevice. Methods for the use of the device for the preparation ofenriched leukocyte populations and cell populations enriched inmonocytes, CD34⁺ hematopoietic stem are precursor cells and the like areprovided. The cell populations enriched in leukocytes and/or monocytesand the like obtained by the use of the devices and methods of thepresent invention can be used to prepare compositions of antigenpresenting cells, e.g., antigen presenting dendritic cells, foradministration to an individual for the induction of an immune response,prepare compositions of pluripotent stem cells, e.g., f-macrophage(f-MΦ), for induction to form epithelial, neuronal, endothelial, orhepatocyte cells, prepare compositions comprising enriched numbers ofhematopoietic stem or precursor cells, and the like.

A tangential flow filtration device of the present invention comprises aremover unit having a cross-flow chamber, a filtrate chamber and afilter disposed therebetween. The filter is in fluid communication onone side, the retentate surface, with the cross-flow chamber, and on theother side, the filtrate surface, with the filtrate chamber. Thecross-flow chamber has an inlet adapted to introduce a sample of bloodconstituents comprising leukocytes into the cross-flow chamber andparallel to the retentate surface of the filter. An outlet is alsoprovided in the cross-flow chamber centrally disposed in a portion ofthe chamber opposite the retentate surface of the filter. The filtersuitable for use in the tangential flow filtration device typically hasan average pore size ranging from about 1 to about 10 microns. Incertain embodiments the filter has an average pore size of about 3 toabout 7 microns, or about 3 to about 5.5 microns.

Further, the device can comprise a means for providing a predeterminedinput rate of the sample into the inlet of the cross-flow chamber and ameans for controlling a filtration rate of filtrate through the filterand into the filtrate chamber. The filtration rate controlling meanslimits the rate of filtration to less than the unopposed filtration ratefor the filter. The sample comprising blood constituents can be providedby a source device such as a leukopheresis device or a containercomprising a sample collected from, for example, a leukopheresis device,and the like.

The tangential flow filtration device can further comprise a recoveryunit. The recovery unit comprising an inlet and an outlet can beinterconnected in a loop format with the cross-flow chamber of theremover unit. In this embodiment of the device, the cross-flow chamberinlet is in fluid communication with the recovery unit outlet, and thecross-flow chamber outlet is in fluid communication with the recoveryunit inlet. The recovery unit can further comprise a sample inlet and awash inlet. In certain embodiments of the tangential flow filtrationdevice the sample inlet and wash inlet are a single shared inlet.Typically, the wash inlet is in fluid communication with a source ofreplacement or wash fluid. The replacement or wash fluid can be, forexample, an isotonic buffer or tissue culture media.

The sample inlet of the recovery unit is in fluid communication with asource of blood constituents. In one embodiment of the present inventionthe source of blood constituents is a cell-processing device. The cellprocessing device can be a leukopheresis device or a device that iscapable of producing a cell population partially enriched forleukocytes. In one example, the cell processing device comprises avessel having a first port and a second port, a monocyte dendritic cellprecursor adhering substrate in fluid communication with the first portand the second port, a screen for retaining the substrate in the vesseland with a pore size sufficient to allow the passage of monocyticdendritic cell precursors and dendritic cells. The device furthercomprises a drain line in fluid communication with the first or secondport and a collection line in fluid communication with the first and orsecond port which is also in fluid communication with the sample inletof the recovery unit of the tangential flow filtration device.

The cell processing device can also comprise a plurality of fluidsources for providing binding media, washing buffer and elution buffer.The device can further comprise a pump for transferring the variousfluids into and out of the cell processing device. A temperaturecontrolling means, such as a heater or cooling device, can also beprovided. In one embodiment of the present invention providing a closedsystem, a blood sample or blood product preparation is provided to thecell processing device comprising a bead material capable of adheringmonocytic dendritic cell precursors. The blood sample is allowed tocontact the bead material for a sufficient time to adhere the monocyticdendritic cell precursors and the device is washed of the other cellcomponents through the drain line. The elution buffer is added to thecell processing device and the monocytic dendritic cell precursors areaseptically passed through the collection line into the sample inlet ofthe recovery unit for further enrichment of the blood sample ofmonocytes.

In one embodiment of the present invention, a tangential flow filtrationdevice for enriching a sample of blood constituents for leukocytes isprovided comprising a remover unit comprising a cross-flow chamber and afiltrate chamber separated by a filter, wherein the cross-flow chamberhas an inlet and an outlet, the outlet centrally disposed in an upperportion of the chamber, and wherein the inlet is disposed above thefilter and introduces fluid into the cross-flow chamber substantiallyparallel to the filter; a means for providing a predetermined input rateof the sample through the cross-flow chamber inlet; and a means forreducing a filtration rate through the filter; wherein the filter has apore size of about 3 microns to about 7 microns; and whereby the sampleis enriched for leukocytes in a retentate in the cross-flow chamber.Further, in another particular embodiment, the filter has a pore size ofabout 3 to about 5.5 microns to enrich the cell population for CD34⁺leukocytes.

In another embodiment of the present invention, a tangential flowfiltration device for enriching a sample of blood constituents formonocytes is provided comprising a remover unit comprising a cross-flowchamber below a filtrate chamber and separated by a filter, wherein thecross-flow chamber has an inlet and an outlet, the outlet centrallydisposed in a lower portion of the chamber, and wherein the inlet isdisposed below the filter and introduces fluid into the cross-flowchamber substantially parallel to the filter; a means for providing apredetermined input rate of the sample through the cross-flow chamberinlet; and a means for maintaining a filtration rate through the filter;wherein the filter has a pore size of about 3 microns to about 7microns; and whereby the sample is enriched for leukocytes in aretentate in the cross-flow chamber. Further, in another particularembodiment, the filter has a pore size of about 3 to about 5.5 micronsto enrich the cell population for CD34⁺ leukocytes.

In yet another embodiment of the present invention, a tangential flowfiltration device for enriching a sample comprising blood constituentsis provided comprising a remover unit (1) having a cross-flow chamber(3) and a filtrate chamber (4) separated by a filter (5), the cross-flowchamber having an inlet (6) and an outlet (7), the outlet disposed abovethe inlet and centrally disposed in an upper portion of the chamber, andwherein the filter is disposed below and substantially parallel to thecross-flow chamber inlet. The device further comprises a means forproviding a predetermined input rate of the sample through thecross-flow chamber inlet; a means for providing a predeterminedfiltration rate of the fluid through the filter, wherein thepredetermined filtration rate is about one-fifth to about oneone-hundredth of the predetermined input rate; and a means for providinga predetermined concentration of blood cells in the sample, wherein thepredetermined concentration of blood cells is typically about 10⁷ toabout 10¹⁰ cells per milliliter. Typically, the filter has a pore sizeof about 3 microns to about 7 microns. Further, in another particularembodiment, the filter has a pore size of about 3 to about 5.5 micronsto enrich the cell population for CD34⁺ leukocytes.

The present invention also provides methods for separating leukocytesfrom a sample of blood constituents comprising leukocytes. In themethods step are provided comprising: (1) introducing the sample into aremover unit through an inlet in the remover unit; (2) subjecting thesample to cross-flow substantially parallel to a filter having a poresize of about 1 to about 10 microns; (3) subjecting the fluid tofiltration through the filter; and (4) selectively removingnon-leukocyte blood constituents from the sample to form a cellpopulation enriched for leukocytes. The sample can be subjected to apartial purification or enrichment by leukopheresis, densitycentrifugation, differential lysis, filtration, or preparation of abuffy coat, for introduction in the remover unit. In one embodiment, thesample is induced to flow with a vortex motion in the cross-flowchamber. Additionally, the cell population enriched for leukocytes canbe washed with a wash solution.

In the methods of the present invention the non-leukocyte bloodconstituents removed from the sample include plasma and platelets,erythrocytes, and the like. The leukocytes of the product from themethods of the invention can comprise a substantially enrichedpopulation of monocytes. The enriched cell population can comprise atleast about 20% leukocytes, but typically comprises at least about 60%or more leukocytes. In one embodiment of the method of the presentinvention steps (1), (2), and (3) are repeated at least two times toform cell population enriched for leukocytes. The cell populationenriched for leukocytes can further be used for the preparation ofmonocytic dendritic cell precursors. In one embodiment the enriched cellpopulation is produced by a method comprising contacting a monocyticdendritic cell precursor adhering substrate with the cell populationenriched for leukocytes; allowing monocytic dendritic cell precursors inthe cell population to reversibly adhere to the substrate to formcomplexes comprising monocytic dendritic cell precursors and substrate;separating the complexes from the non-adhering leukocytes to obtaincomplexes comprising monocytic dendritic cell precursors; and culturingthe monocytic dendritic cell precursors to differentiate the precursorsto form immature or mature dendritic cells. In one particular embodimentthe monocytic dendritic cell precursors are eluted from the substrateprior to culturing. The substrate for adhering the monocytic dendriticcell precursors can comprise glass, polystyrene, plastic, orglass-coated polystyrene microbeads.

In still another embodiment of the present invention a method forenriching a sample of blood constituents for leukocytes is providedcomprising: (1) introducing the sample into a tangential flow filtration(TFF) unit, the TFF unit comprising a cross-flow chamber, a filtratechamber, and a filter in fluid communication with the cross-flow chamberand the filtrate chamber, the filter having a pore size of about 1 toabout 10 microns; (2) recirculating the sample through the TFF unit at apredetermined input rate and a predetermined filtration rate, thepredetermined input rate at least five times the predeterminedfiltration rate; wherein the predetermined filtration rate is less thanthe unopposed filtration rate for the filter; and (3) isolating a cellpopulation enriched for leukocytes. The method can result in an enrichedcell population that is substantially free of non-leukocyte bloodconstituents including plasma, platelets and erythrocytes. The enrichedcell population produced by this method can comprise at least about 20%leukocytes, and typically at least about 60% or more leukocytes. Themethod can further comprise the collecting of blood from a subject andpreparing the sample from the blood by leukopheresis, densitycentrifugation, differential lysis, filtration, or preparation of abuffy coat.

Once the cell population has been enriched for leukocytes the method canfurther comprise preparing a particular cell type that can be inducedfrom the leukocytes, such as, for example, dendritic cell precursors,CD34⁺ hematopoietic stem cells, or pluripotent stem cells, such as,f-macrophage, and the like. In one particular embodiment dendritic cellscan be prepared from the enriched cell population. In this method thedendritic cells are prepared by: contacting a monocytic dendritic cellprecursor adhering substrate with the enriched cell population; allowingmonocytic dendritic cell precursors in the enriched cell population toreversibly adhere to the substrate to form complexes comprisingmonocytic dendritic cell precursors and substrate; separating thecomplexes from the non-adhering leukocytes to obtain complexescomprising monocytic dendritic cell precursors; and culturing themonocytic dendritic cell precursors to differentiate the precursors toform immature or mature dendritic cells. The substrate can compriseglass, polystyrene, plastic or glass-coated polystyrene microbeads.Additionally, the monocytic dendritic cell precursors can be culturedwith cytokines that promote the differentiation of monocytes intodendritic cells. In a particular embodiment the cytokines are GM-CSF andIL-4. Further, the dendritic cells can be matured to mature dendriticcells.

Once the dendritic cell precursors have been isolated, the dendriticcells can be cultured with an antigen under conditions conducive forprocessing the antigen to form antigen loaded dendritic cells. Theantigen loaded dendritic cells can then be administered to an individualor the antigen loaded dendritic cells can be cultured in vitro or exvivo with T cells to induce the formation of antigen specific cytotoxicT cells. The cytotoxic T cells can be administered to an individual inneed of an induced antigen specific immune response, such as in thetreatment of cancer and bacterial or viral infection.

A cell population enriched for hematopoietic stem cells can be produced.In one embodiment, an individual can be provided with a stem cellmobilizing agent, such as for example, G-CSF, GM-CSF, AMD3100 (or otheragents that inhibit CXCR-4 function), or high- or low-dosecyclophosphamide, and the like. The stem cell mobilizing agent inducesthe proliferation of CD34⁺ stem cells which are released into theperipheral blood steam. A leukapheresis sample from the individual isintroduced into a tangential flow filtration (TFF) unit, the TFF unitcomprising a cross-flow chamber, a filtrate chamber, and a filter influid communication with the cross-flow chamber and the filtratechamber, the filter having a pore size of about 3 to about 5.5 microns;(2) recirculating the sample through the TFF unit at a predeterminedinput rate and a predetermined filtration rate, the predetermined inputrate at least five times the predetermined filtration rate; wherein thepredetermined filtration rate is less than the unopposed filtration ratefor the filter; and (3) isolating a cell population enriched for CD34⁺leukocytes. The method can result in an enriched cell population that issubstantially free of non-leukocyte blood constituents including plasma,platelets and erythrocytes. The enriched cell population produced bythis method can increase the percentage of CD34⁺ cells 2 to 5 fold from1% to about 5% of leukaphoresis material to about 10% to about 40% ofthe cells in an enriched cell population.

Further, monocytes isolated as described above can be cultured in M-CSFcontaining medium in a non-adhesive cell culture container, e.g., aTeflon culture bag. Culture of the monocytes in M-CSF results in theproduction of a substantial number of CD34⁺ cells. Cell populationsenriched in leukocytes or monocytes as described above can also becultured in the presence of a number of other cytokines and leukokinesknown in the art to induce the production of a number of otherprogenitor cell types. for example, the cell population enriched inleukocytes and/or monocytes can be cultured in the presence of VEGF,bFGF, IGF-1, EGF and fetal serum on a fibronectin coated surface anddiscarding non-adherent cells to obtained endothelial-like circulatingangiogenic cells, or f-MΦ can be differentiated into epithelial cells byculturing in EGF, differentiated into neuronal and endotherlial cells byincubation in NGF or VEGF respectively or differentiated intohepatocytes by incubating in the presence of HGF, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1C depict embodiments of the tangential flow filtrationdevice for the separation of leukocytes and also monocytes from a bloodproduct-sample. FIG. 1A provides an embodiment of the device for theenrichment of leukocytes wherein the cross-flow chamber is above thefiltration chamber. FIG. 1B depicts a front view of the device whereinthe input of sample is below the filter and the filtrate passing upwardthrough the filter for the enrichment of monocytes. FIG. 1C is anoverhead view of the device depicted in FIG. 1B.

FIG. 2 depicts an example of tangential flow filtration (TFF) performedon samples of leukopheresis product under various conditions. Samples of10 ml of leukopheresis product, diluted 1:5 in a buffer ofPBS+heparin+DNase I were subjected to TFF using a 3 micron filter with afiltration rate of 15 ml/min. The percentage of leukocytes (WBC) in theretentate (designated “Retentate”; hatched bars) and filtrate(designated “Filtrate”; dark bars) after TFF are shown. Therecirculation (input) rates for Max, 10, 9, 8, 7, and 6 corresponded to1680, 1380, 1080, 870, and 540 ml/min, respectively.

FIG. 3 depicts the results of a study of TFF performed on leukopheresisproduct in a TFF device using a 3 micron filter, with a recirculation(input) rate of 1080 ml/min at three different filtration rates (11, 15,and 19.6 ml/min). The percentage of leukocytes (designated “WBC”) in theretentate (hatched bars) or filtrate (designated “Filtrate”; dark bars)is shown.

FIG. 4 depicts additional results of TFF performed on samples ofleukopheresis product for the study described in FIG. 1. Samples of 10ml of leukopheresis product, diluted 1:5, were subjected to TFF using a3 micron filter with a filtration rate of 15 ml/min. The percentage oferythrocytes (designated “RBC”) in the retentate (hatched boxes) andfiltrate (dark boxes) after TFF are shown. The recirculation (input)rates for Max, 10, 9, 8, 7, and 6 corresponded to 1680, 1380, 1080, 870,and 540 ml/min, respectively.

FIG. 5 depicts additional results of TFF performed on samples ofleukopheresis product for the study described in FIG. 2. Samples of 10ml of leukopheresis product, diluted 1:5, were subjected to TFF using a3 micron filter, with a recirculation (input) rate of 1080 ml/min atthree different filtration rates (11, 15, and 19.6 ml/min). Thepercentage of erythrocytes (designated “RBC”) in the retentate (hatchedbars) or filtrate (designated “Filtrate”; dark bars) is shown.

FIG. 6 depicts an example of the effects of increasing the concentrationof leukopheresis material in the sample. 50 ml of leukopheresismaterial, diluted 1:5 in PBS+heparin+DNase I, was subjected to TFF usinga device having a 3 micron pore size filter. The percentage oferythrocytes (designated “RBC”) and leukocytes (designated “WBC”) in theretentate (designated “Retentate”) is shown as a function of thefiltration rate.

FIG. 7 depicts an example of the separation of leukopheresis productbetween 3 micron and 5 micron filters upon scale-up of leukopheresisproduct (120 ml or ½ of an entire unit). The recirculation (input) ratewas 1680 ml/min, and the filtration rate was 15 ml/min. For TFFperformed using a 5 micron filter, about 80% of the erythrocytes(designated “RBC”; dark shaded bars) were removed from the retentate,while about 62% of input leukocytes (designated “WBC”; light hatchedbars), or greater than about 70% of input monocytes, were retained. Incontrast, using the 3 micron filter, about 65% of input leukocytes wereretained in the retentate, but only 3% of the erythrocytes were removed.

FIG. 8 depicts a comparison of the separation of leukopheresis productthrough an approximately 4.5 micron and 8 micron filters upon scale-upof the quantity of leukopheresis product provided as the sample. Therecirculation (input) rate was 1680 ml/min, the filtration rate was 15ml/min. For TFF performed using a 4.5 micron filter, about 99% of theerythrocytes (designated “RBC”; dark shaded bars) were removed from theretentate, while about 90% of input leukocytes (designated “WBC”; lighthatched bars) were retained. In contrast, the 8 micron filter, about 98%of input erythrocytes were removed, but only 4% of the leukocytes wereretained.

FIG. 9 depicts an example of the separation on 4.5 micron filters uponscale-up of leukopheresis product processed (250 ml or an entire unit).The recirculation (input) rate was 1680 ml/min, and the filtration ratewas 15 ml/min. For TFF on 4.5 micron filters performed for 90 min onthree different leukopheresis products, between 80 to 95% of theerythrocytes were removed from the retentate, while about 80 to 100% ofinput monocytes were retained. Following TFF of one leukopheresissample, the retentate was also assayed and found to have about only 2%of the input platelets and 3% of the input plasma (designated Exp 7 inTable 1).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides devices and methods for processing aheterogenous mixture of blood constituents to provide an enrichedpopulation of leukocytes. In one aspect of the invention, devices andmethods are provided for the enrichment of leukocytes by the selectiveremoval of non-leukocyte blood constituents, e.g., plasma, plateletsand/or erythrocytes, and the like. In another aspect, devices andmethods are provided for the enrichment of monocytes by the selectiveremoval of non-monocyte blood constituents, including, for example, theremoval of lymphocytes, erythrocytes, platelets and the like from themixture.

An enriched population of leukocytes is typically prepared from asample, or fluid mixture, comprising blood constituents. The term “bloodconstituents” as used herein refers to any material typically present inblood, including such material typically present in diseased as well asnon-diseased states. Blood constituents include leukocytes and caninclude, for example, lymphocytes, monocytes, erythrocytes, neutrophils,eosinophils, natural-killer (NK) cells, and/or platelets, soluble orinsoluble protein or protein complexes (e.g., enzymes, immunoglobulins,or immunoglobulin-antigen complexes), other macromolecular componentssuch as, e.g., lipids, or any other portion of whole blood that can bephysically separated, irrespective of its precise molecular or cellularmakeup, including, e.g., plasma or serum.

The sample, or fluid mixture, can be partially enriched for leukocytesprior to carrying out the methods of the present invention. The term“leukocyte” is used interchangeably with the term “white blood cells”(“WBCs”). These terms include mononuclear agranulocytes, which include,e.g., monocytes, dendritic cell precursors, and lymphocytes, as well aspolymorphonuclear granulocytes with segmented nuclei and cytoplasmicgranules, including neutrophils, eosinophils, basophils, and mast cells.“Monocyte” refers to a class of myeloid-derived leukocytes, generallylarger than lymphocytes, with an ovoid or kidney-shaped nucleus,typically containing lysosomal granules and typically expressing CD14.

In certain aspects of the present invention, lymphocytes are separatedfrom the leukocytes. “Lymphocyte” refers to cells derived from lymphoidprogenitor cells and includes B-lymphocytes, T-lymphocytes, andnatural-killer (NK) cells. The term “small lymphocytes” refers tolymphocytes that are about 7-8 microns in diameter.

As used herein, the term “population of leukocytes” refers to any groupof cells that includes leukocytes. A population of leukocytes caninclude a broad range of leukocyte sub-types or of particular sub-types,such as, e.g., monocytes and/or monocytic dendritic precursor cells. Theterms “enrichment”, “enrich” and “enriched” mean that the processing ofa mixture of blood constituents using the devices, or following themethods of the present invention results in a cell population having ahigher percentage of viable leukocytes, in relation to otherconstituents, than the initial mixture (i.e., prior to enrichment). Asused herein, the term “viable” refers to a leukocyte that is capable ofdifferentiation under suitable culture conditions.

The devices according to the present invention utilize tangential flowfiltration to enrich for a population of leukocytes. The terms“tangential flow filtration” and “cross-flow filtration” are usedinterchangeably and refer to the separation of suspended particles(e.g., cells) from a fluid mixture, including the separation ofparticles of a defined characteristic (e.g., a desired size range) froma heterogeneous mixture of particles in the fluid mixture. The particlesare separated by passing or circulating the fluid mixture (e.g., asample fluid) in a sample chamber substantially parallel or tangentialto a filter (e.g. the surface of the filter facing the sample fluid),typically under some positive pressure, with the fluid mixturecomprising the concentrated particles, or leukocytes, continuing to flowtangential to the membrane surface.

Generally, determination of which particles are removed in the“filtrate,” i.e., that portion of fluid passing through the filter, andthose particles retained in the “retentate” is dependent on a variety offactors. Such factors include, e.g., filter pore size, input rate,filtration rate, concentration of particles in the fluid mixture,temperature, and viscosity of the fluid mixture. As used herein, “poresize” refers to the average size of the pores in the filter. “Inputrate” refers to the rate at which a sample (e.g., fluid mixture) isintroduced into the chamber housing the filter. Where the sample isrecirculated multiple times across a filter (e.g., in a device accordingto the present invention), the input rate is also referred to as the“recirculation rate.” “Cross-flow” refers to the substantially parallel(i.e., parallel to the surface of the filter in any direction) flow ofthe fluid mixture across the filter. “Cross-flow rate” refers to therate of flow of sample, or fluid mixture, over and substantiallyparallel to the filter; the cross-flow rate of the fluid mixture isgenerally dependent on a variety of parameters, including, for example,the input rate and the size and shape of the chamber housing the filter.“Filtration rate” refers to the rate of flow of the fluid mixturethrough the filter. The filtration rate for a device and the methodsaccording to the present invention is typically less than the unopposed(i.e., open tube) filtration rate. “Output rate” refers to the rate ofremoval of the fluid mixture from the cross-flow chamber, other than thefluid mixture passing through the filter (i.e., the filtrate). Theoutput rate is generally equal to the input rate minus the filtrationrate.

As used herein, the term “filter” refers to any article made of anymaterial or combination of materials having a plurality of pores thatallow one or more components (e.g., blood constituents) of a sample orfluid mixture subjected to cross-flow across the article to pass throughit, thereby separating those components (e.g., non-leukocytes) fromother components (e.g., leukocytes). The surface of a filter can haveany suitable area, such as, for example, about 42 to about 145 mm indiameter, although filters of greater and lesser area can be used. Incertain embodiments, only one filter is used in a TFF device. In otherembodiments, additional filters can be used in a TFF device.

The filter typically employed in the TFF device of the present inventioncan be chosen from a wide range of organic polymeric filters. Suchfilters include, but are not limited to, microporous membranes of nylon,polyvinylidene fluoride (PVDF), cellulose acetate/nitrate, polysulfone,polycarbonate, polyethylene, polyester, polypropylene, and polyamide.Other filters, such as ceramic filters and metallic filters, can also beused. Both hydrophilic and hydrophobic, charged and uncharged filterscan be used. In certain applications, hydrophilic filters can bepreferred.

A filter of the present invention typically comprises a number of poresdistributed across the area of the filter. In certain embodiments, thefilter has a pore size with a small variation in pore size. For example,the variability in the pore size can be about ±20%, or within the rangeof about +0 to about 20%. In a typical embodiment, “nuclepore” or “tracketched” filters are used (e.g., Poretics® polyethylene or polycarbonatetrack-etched filter membranes (Osmonics, Minnetonka, Minn.)). Thesefilters typically have a smooth surface with tightly controlled poresizes in the material. Such filters are typically prepared by exposing aflat sheet of non-porous plastic to a source of radioactive particles,which are energetic enough to pierce to plastic sheet. The “tracks” arethen enlarged in diameter by exposure to chemical solvents or etchingagents. The size of the pores can be controlled by the track etchingconditions.

The present invention takes advantage of differences between variouscell types in blood to enrich for leukocytes (e.g., monocytes, dendriticcell precursors, and the like). Such differences can include, e.g.,differences in size, shape and/or deformability. The size anddeformability of cells in human blood typically varies by cell type.Erythrocytes (red blood cells) are typically biconcave disk shaped,enucleate, measure about 7 microns in the major diameter and arerelatively deformable. Polymorphonuclear leukocytes cells are typicallyspheroidal, also about 7 microns, but less deformable than erythrocytes.Of the mononuclear cells, lymphocytes are typically 7 to 10 microns, andmonocytes usually are in the range of 10 to 15 microns.

In various embodiments, the filter pore size is selected to enrich forleukocytes, and/or to fractionate blood constituents, thereby enrichingfor leukocytes. For example, in certain embodiments, monocytes having anominal diameter of 10 to 15 microns, and erythrocytes having a nominaldiameter of 7 microns, can be separated by TFF using a filter having apore size of about 5 to about 5.5 microns. In a particular embodiment afilter of 4.5 microns was used to successfully separate monocytes fromthe other cellular constituents of a leukopheresis sample.

In other embodiments, the filter pore size can be within the range ofabout 1 to about 10 microns, or about 3 to about 8 microns, or about 3to about 5 microns. A filter pore size in the range of about 3 micronscan retain most leukocytes, and effect less efficient removal oferythrocytes from the leukocytes. In contrast, a filter pore size in therange of about 8 microns can effect more efficient removal oferythrocytes, but increases the loss of leukocytes in the filtrate. Afilter size of about 3 to about 5.5 microns can be used to enrich forCD34⁺ hematopoietic stem cells.

The enrichment of leukocytes from other cellular blood constituents canalso be affected by the input rate, the filtration rate, and/or theconcentration of cells in the sample or fluid mixture. For example,erythrocytes are more deformable than leukocytes and can, therefore, bemore readily passed through a filter pore size smaller than the majordiameter of the erythrocytes (e.g., less than about 7 microns). In aspecific example, erythrocytes can be separated from leukocytes usingfilters having pore size of about 5 microns. In other embodiments, thefilter pore size is decreased to about 3 microns, and the concentrationof cells increased (supra) to efficiently separate erythrocytes fromleukocytes.

The enrichment of leukocytes from other cellular blood constituents canalso be effected by maintaining a filtration rate that is less than theunopposed (i.e., open tube) filtration rate under the same input orrecirculation rate. In other embodiments, the loss of leukocytes to thefiltrate can be reduced by maintaining an input or recirculation ratethat is greater than the filtration rate. In exemplary embodiments, theinput or recirculation rate can be at least about five time, at leastabout 10 times, at least about 20 times, at least about 50 times, or atleast about 100 times, the filtration rate.

A sample, or fluid mixture, comprising various blood constituents forcell fractionation by TFF can be obtained from a variety of sources andcan include fluid mixtures of blood products at any of the variousstages of processing. For example, blood sources can be either human ornon-human. In addition, fluid mixtures can be, for example, whole blood,various dilutions of whole blood, or whole blood or blood dilution thathas been subjected to processing by, e.g., removal of plasma or otherblood constituents. Thus, the fluid mixture can include, for example, ablood cell population that is already at least partially enriched forleukocytes.

Blood constituents, or populations of leukocytes, can be prepared bymethods known to those skilled in the art. Such methods typicallyinclude collecting heparinized blood, apheresis or leukopheresis,preparation of buffy coats, resetting, centrifugation, density gradientcentrifugation (e.g., FICOLL-HYPAQUE®, PERCOLL®, sucrose, and the like),differential lysis of non-leukocyte cells, filtration, and the like. Aleukocyte population can also be prepared by collecting blood from asubject, defibrinating to remove the platelets and lysing the majorityof red blood cells. The population of leukocytes can optionally beenriched for monocytes by, for example, centrifugation through aPERCOLL® gradient.

The fluid mixture comprising the blood constituents can optionally bediluted or concentrated, as desired. For example, in certainembodiments, the blood constituents are diluted 1:2, 1:5, 1:10, or anyother suitable dilution. Blood constituents can be diluted in, forexample, isotonic buffers (e.g., PBS or HEPES-buffered saline), tissueculture media and the like. Typically, the sample of blood constituentssubjected to TFF has a cell concentration of about 10⁶ to about 10⁸cells per ml.

Blood cell populations can be obtained from a variety of types ofsubjects, according to the desired use of the enriched population ofleukocytes. The subject can be a healthy subject. Alternatively, bloodcells can be obtained from a subject in need of immunostimulation, suchas, for example, a cancer patient or other patient for whichimmunostimulation may be beneficial. Likewise, blood cells can beobtained from a subject in need of immune suppression, such as, forexample, a patient having an autoimmune disorder (e.g., rheumatoidarthritis, diabetes, lupus, multiple sclerosis, and the like). A bloodcell population also can be obtained from an HLA-matched healthyindividual for administration to an HLA-matched patient in need ofimmunostimulation. A blood cell population can also be collected from anindividual that has been administered a stem cell mobilization agentsuch as for example GM-CSF, G-CSF, AMD3100 (or other agent that inhibitsCXCR-4 function), or low- or high-dose cyclophosphamide (Deliliers etal., Leuk. Lymphoma 43:1957, 2002) and the like. The individual can be apatient that wilt received enriched cell population, a relative, or aHLA-matched individual.

In certain embodiments, the enriched population of leukocytes can becollected in the retentate, while other blood constituents pass into thefiltrate. For example, for enrichment of a population of leukocytes(e.g., including monocytes and lymphocytes), other blood constituentssuch as plasma, platelets, and/or erythrocytes can be among theconstituents selectively removed into the filtrate. In additionalembodiments, lymphocytes, or small lymphocytes, can be selectivelyremoved and passed into the filtrate.

The devices according to the present invention as depicted in FIGS. 1Athrough 1C typically comprise a cross-flow chamber (3) and a filtratechamber (4). A filter (5) is positioned between and with one surface influid communication with the cross-flow chamber (the retentate surface)and other surface in fluid communication with the filtrate chamber (thefiltrate surface). The cross-flow chamber, filtrate chamber and filtercomprise a remover unit (1). In one embodiment, the cross-flow chambertypically has a volume of about 55 ml, and the filtrate chamber has avolume of about 25 ml. The filter diameter is typically substantiallythe same as the diameter of the cross-flow chamber. In certainembodiments used to demonstrate the utility of the present invention,the filter is about 140 mm to about 143 mm in diameter.

The fluid mixture enters the cross-flow chamber (3) through a fluidinlet (6) that is typically situated adjacent to the retentate surfaceof the filter and such that the fluid mixture (e.g., sample) enters thechamber substantially parallel to the retentate surface of the filter.Typically, fluid is removed from the cross-flow chamber (3) through afluid outlet (7), which is usually located at a portion of a cross-flowchamber perpendicular to the retentate surface of the filter. In certainexemplary embodiments, the cross-flow chamber inlet (6) diameter isabout 7 mm to about 8 mm, and cross-flow chamber outlet (7) diameter isabout 8 mm to about 10 mm. The filtrate is removed through an outlet (8)in the filtrate chamber (4).

Typically, the fluid mixture is introduced into the cross-flow chamberat a sufficient input rate such that the cross-flow of the fluid mixtureacross the surface of the filter (retentate surface) is at a velocityhigh enough to gently disrupt and back-mix fluid and cells at thecontact surface of the filter, i.e., the boundary layer. As used herein,“boundary layer” refers to that layer of fluid adjacent to and on theretentate side of the filter, typically left by fluid passing throughthe filter. This disruption of the boundary layer facilitates efficientfiltration by preventing the material at the contact surface of thefilter from binding to the filter or becoming stagnant, which can hinderefficient filtration. The input rate of the fluid mixture is usually notsufficient, however, to cause lysis of a substantial number ofleukocytes.

In certain embodiments, the blood constituents are passed across theretentate surface of the filter by pumping the fluid mixture into thecross-flow chamber (3). The pump used to drive the cross-flow of fluidacross the filter is referred to as the “cross-flow pump” or“recirculating pump” (14). The cross-flow pump can include any pumpingdevice in fluid communication with the cross-flow chamber (3) sufficientto introduce the flow of fluid into the chamber and across the filter atthe specified input rate, without causing substantial damage to thecells (e.g., cell lysis). A cross-flow pump suitable for use in thepresent invention can include, e.g., a peristaltic pump, piston pump,diaphragm pump, or roller pump. A peristaltic pump can be used, forexample, where it is desired to maintain the TFF device as part of a“closed” system.

The fluid mixture is typically pumped into the cross-flow chamber (3) atan input rate that exceeds the filtration rate. In an exemplaryembodiment, the input rate is about 1680 ml/minute, and the filtrationrate is about 15 ml/minute. In other exemplary embodiments, the inputrate is about 1600 to about 1800 ml/minute, and the filtration rate isabout 10 to about 20 ml/minute. Non-leukocytic material (e.g.,erythrocytes, immune complexes, proteins, and the like) pass through thefilter (5) into a filtrate chamber (4).

As discussed supra, the filtration rate is typically less than theunopposed (i.e., open tube) rate. The filtration rate can be controlled,for example, by reducing or restricting the size of the filtrate chamberoutlet, by use of a second pump means (e.g., a “filtration pump”) torestrict the flow, and the like.

In another exemplary embodiment, the introduction of a fluid mixtureinto the device creates a vortex motion within the fluid. This can bedone, for example, by introducing the fluid mixture, for examplesubstantially parallel to a circular filter in a cylindrical cross-flowchamber, at an input rate about 5 or about 10 to about 100 times thefiltration rate. The flow through is removed by means of an outlet (7)located in the cylindrical chamber perpendicular to the filter andtypically adjacent to the center of the filter surface. This arrangementcauses the flow to spiral inward toward the center of the filter. Theflow is typically not turbulent, or at such a high rate, so as to causesubstantial lysis of the leukocytes. As discussed above, the flow canalso “scrub” the filter surface to prevent binding or stagnation at theboundary layer. By calibrating the input rate such that it is large(e.g., at least about 5 times) relative to the filtration rate, theresulting enriched population of leukocytes can be at least about 20, orat least about 40 percent, or more, leukocytes.

In another exemplary embodiment, the retentate is recirculated toincrease efficiency of separation. For example, a fluid mixturecomprising blood constituents can be introduced into the cross-flowchamber, and then retentate can be withdrawn through the fluid outlet(7) in the cross-flow chamber to another chamber, such as, e.g. achamber from which the fluid was initially provided (“a recovery unit”;(2)). The fluid mixture in the recovery unit can then be re-introducedinto the cross-flow unit. By connecting the recovery unit (2) andremover unit (1) in “loop format,” continuous recirculation andfiltration of the fluid mixture can be achieved. Alternatively, theretentate can be withdrawn through the fluid outlet (7) of thecross-flow chamber (3) and directly reintroduced into the cross-chamberinlet (i.e., without passing through a recovery unit or anotherchamber). The fluid mixture can be passed through the cross-flow unitfor any suitable period of time. In certain embodiments, the fluidmixture can be recirculated for about 5 to about 60 minutes, or more, toachieve the desired leukocyte cell purity or enrichment.

In yet another embodiment, the volume of the fluid mixture can beadjusted by adding a buffer, a wash solution or other solution(collectively referred to as a “replacement liquid”). The wash solutioncan, for example, be combined with a fluid mixture in a recovery unit(e.g., through a solution inlet; (13)), in a remover unit, at a pump(14), in tubing extending to or from the remover unit, or at any otherconvenient location. The cells in the retentate can thus be enriched andwashed in the same operation. Typically, the wash solution is isotonicwith the cells. Suitable buffer and wash solutions can include a varietyof buffers (e.g., phosphate-buffered saline (PBS) or HEPES-bufferedsaline), tissue culture media, and the like.

In certain embodiments, cell populations are enriched for a populationof leukocytes in a closed, aseptic system. As used herein, the terms“closed, aseptic system” or “closed system” refer to a system in whichexposure to non-sterile, ambient, or circulating air or othernon-sterile conditions is minimized or eliminated. Closed systems forenriching cell populations generally exclude centrifugation in open toptubes, open air transfer of cells, culture of cells in tissue cultureplates or unsealed flasks, and the like. The entire filtration system,including, e.g., any cell containers, incubators, tissue culturevessels, or other apparatus for cell processing (infra), can bemaintained as a “closed” system. In a typical embodiment, the closedsystem allows aseptic enrichment of leukocytes and, optionally, transferfrom an initial collection vessel to a sealable tissue culture vessel,without exposure to non-sterile air. Typically, a peristaltic pump(FIGS. 1A and 1C; (15)) means is used in a closed system.

In another aspect of the invention, a heterogeneous mixture of bloodconstituents is substantially enriched for leukocytes by the selectiveremoval from the mixture of non-leukocyte blood constituents, including,e.g., plasma, platelets, erythrocytes, and the like. As used herein, theterm “substantially enriched” means that the cell population recoveredin the retentate, following as many cycles of recirculation as desired,is comprised of at least about 20%, or at least about 40%, or at leastabout 60%, of the desired cell type (e.g., leukocytes). In otherembodiments, a heterogeneous mixture of blood constituents is enrichedfor leukocytes to form an enriched population of leukocytes that issubstantially free of non-leukocyte blood constituents. As used herein,the term “substantially free” means that the enriched population ofleukocytes comprises at least 50% leukocytes.

In an exemplary embodiment of this aspect of the present invention, theTFF device comprises a cross-flow chamber (3) with a volume of about 55ml and a filtrate chamber (4) with a volume of about 25 ml. Further thedevice comprised the following: a filter pore size of about 1 to about10 microns, or about 2 to about 8 microns, or about 3 to about 5microns; an input rate of about 1600 to about 1800 ml/min; a filtrationrate of about 12 to about 17 ml/min, and a filter diameter of about 142mm. The initial fluid mixture typically has a cell concentration of atleast about 10⁷ cells per ml (e.g. leukocytes and other cells).

In another aspect of the invention, a heterogeneous mixture of bloodconstituents is substantially enriched for monocytes by the selectiveremoval of non-monocyte blood constituents, including, for example, theremoval of lymphocytes from the mixture. As used herein, the terms“selective removal”, “selectively removed” and “selectively removing”refer to the preferential removal of one cell type and enriching foranother cell type. In an exemplary embodiment of this aspect, the TFFdevice comprises a cross-flow chamber (3) with a volume of about 55 mland a filtrate chamber (4) with a volume of about 25 ml. Further, thedevice comprised the following: a filter pore size of about 1 to about10 microns, or about 2 to about 8 microns, or about 3 to about 5microns; an input rate of about 1600 to about 1800 ml/min; a filtrationrate of about 12 to about 17 ml/min; and a filter diameter of about 142mm. The initial fluid mixture typically has a cell concentration of atleast about 10⁷ cells per ml (e.g., monocytes and other cells). In thisembodiment the device was operated in an inverted manner.

Culture, Expansion and Differentiation of Enriched Cell Populations

Following enrichment of a leukocyte cell population as described, supra,the leukocytes optionally can be cultured to maintain their viability,increase cell numbers and/or differentiate the cells to another celltype. Suitable tissue culture vessels, include, for example, tissueculture flasks, bags, plates, bioreactors (including a fermenter), andthe like.

In an exemplary embodiment, the enriched population of leukocytes can becultured in a closed, aseptic system, such as a bioreactor, tissueculture bag, and the like. The closed system can have an inlet and/oroutlet for the controlled, aseptic introduction or removal of fluids(e.g., tissue culture media, washing buffer), gases, cells, and thelike.

In another exemplary embodiment, an enriched population of leukocytescan be transferred to a bioreactor. The bioreactor can be equipped withappropriate inlets and/or outlets for introducing cells, sterile gas(e.g., oxygen, carbon dioxide, and/or air), tissue culture media, andthe like. The bioreactor can also have means for controlling thetemperature. The bioreactor is typically operated at about 37° C. Thebioreactor also can include means for agitating the cells and/or culturemedium in the bioreactor. The agitation means can include, for example,a paddle or a spin filter (which also can function as an outlet formedia).

In yet another exemplary embodiment, an enriched population ofleukocytes can be transferred to closed system, such as a tissue culturebag. Suitable tissue culture bags include, for example, STERICELL®culture containers (Nexell Therapeutics Inc.) or TEFLON® culture bags(American Fluoroseal Corp.). The closed system can have any suitablesize or volume, as will be appreciated by the skilled artisan. Suitablevolumes include, for example, from about 0.01 liters to about 5 liters,or about 0.01 liters to about 0.05 liters, although greater and lesservolumes are possible and within the scope of the present invention.

In various embodiments according to the present invention, cellpopulations enriched for leukocytes (e.g., including monocytes,monocytic dendritic cell precursors, CD34⁺ hematopoietic stem cells, orother precursor cells) optionally can be cultured and differentiated bythe addition of an appropriate inducing agent to obtain cells of aparticular cell type, including for example, immature or maturedendritic cells f-macrophage, CD34⁺ hematopoietic stem or precursorcells or other precursor cells. Suitable tissue culture media include,for example, AIM-V, RPMI 1640, DMEM, X-VIVO 15™, and the like. Thetissue culture medium can be supplemented, as desired, with amino acids,vitamins, cytolines, such as granulocyte/macrophage colony stimulatingfactor (GM-CSF) and/or interleukin 4 (IL-4), divalent cations, and thelike, to promote differentiation of the cells to, immature dendriticcells, for example. A typical cytokine combination is about 500 units/mleach of GM-CSF and IL-4.

The enriched population of leukocytes can be cultured for any suitabletime. In certain embodiments, suitable culture times for thedifferentiation of cells to immature dendritic cells can be about 4 toabout 7 days. The differentiation of immature dendritic cells fromprecursors can be monitored by methods known to those skilled in theart, such as by the presence or absence of cell surface markers (e.g.,CD14⁻, CD11c⁺, CD83^(lo), HLA-DR⁺). Immature dendritic cells can also becultured in appropriate tissue culture medium to expand the cellpopulation and/or maintain the immature dendritic cells in a state forfurther differentiation or antigen uptake, processing and presentation.For example, immature dendritic cells can be maintained in the presenceof GM-CSF and IL-4.

In certain embodiments, immature dendritic cells are preferred foroptimal antigen presentation because they retain the ability to processnew antigen (See, e.g., Koch et al., J. Immunol. 155: 93-100, 1995.) Incontrast, mature dendritic cells (e.g., CD14⁻, CD11c⁺, CD83⁺, CD86⁺,HLA-DR⁺), those that have been exposed to and process antigen and tosuitable maturation agents, have typically lost the ability toefficiently process new antigens.

During culture, immature dendritic cells can optionally be exposed to apredetermined antigen. Suitable predetermined antigens can include anyantigen for presentation to T-cells (e.g., for activation, stimulationof proliferation, induction of anergy, and the like). In one embodiment,immature dendritic cells are cultured in the presence of a tumorassociated antigen, such as, for example, prostate specific membraneantigen (PSMA) (e.g., for cancer immunotherapy and/or tumor growthinhibition). Other antigens can include, for example, bacterial andviral antigens, tumor specific or tumor associated antigens (e.g., tumorcell lysate, tumor cell membrane preparation, isolated antigens fromtumors, fusion proteins, liposomes, and the like), and any otherantigen. Following contacting with antigen, the cells can be culturedfor any suitable time to allow antigen uptake and processing, to expandthe population of antigen-specific dendritic cells, and the like.Immature dendritic cells can also be matured into mature dendritic cellsthat present antigen in the context of MHC molecules. Such maturationcan be performed, for example, by culture in the presence of maturationfactors, such as cytokines (e.g., TNF-α), bacterial products (e.g.,BCG), and the like.

In yet another aspect of the invention, a heterogeneous mixture of bloodconstituents is substantially enriched for monocytic dendritic precursorcells. Following enrichment of a population of cells for leukocytes ormonocytes, as described supra, monocytic dendritic cell precursors, suchas those from peripheral blood, can be isolated from the enrichedpopulation through selective adherence to a substrate (e.g., a monocyticdendritic cell precursor binding substrate). Such a substrate can beprovided by, for example, a tissue culture dish or flask. Alternatively,a substrate having a high surface area to volume ratio, such as aparticulate or fibrous substrate, as disclosed in PCT/US02/23865, filed25 Jul. 2002, the disclosure of which is incorporated by referenceherein), can be used. The monocytic dendritic cell precursors can bemonocytes that selectively adhere to the substrate to form complexes ofmonocytic dendritic cell precursors and substrate, while otherleukocytes remain unbound (“non-adhering”). The bound leukocytes arethen separated from the unbound leukocytes to form a population of cellsenriched in monocytic dendritic cell precursors on the substrate. Themonocytic dendritic cell precursors can be cultured and differentiatedon the substrate, or eluted from the substrate and then cultured anddifferentiated separately, to obtain immature and/or mature,antigen-presenting dendritic cells. In accordance with this aspect, themonocytic dendritic cell precursors optionally can be isolated anddifferentiated in a closed, aseptic system.

According to another aspect, dendritic cells exposed to a predeterminedantigen can be used to activate T cells in vitro or in vivo against theantigen. The dendritic cells optionally can be used immediately afterexposure to antigen to stimulate T cells. Alternatively, dendritic cellscan be maintained in the presence of a combination of cytokines (e.g.,GM-CSF and IL-4) prior to exposure to antigen and T cells. In a specificembodiment, human dendritic cells are used to stimulate human T cells invitro or in vivo.

T cells or a subset of T cells can be obtained from various lymphoidtissues. Such tissues include but are not limited to the spleen, lymphnodes, and peripheral blood. T cell purification can be achieved, forexample, by positive or negative selection including, but not limitedto, the use of antibodies directed to CD2, CD3, CD4, CD5, and/or CD8.

T cells can be co-cultured with dendritic cells exposed to thepredetermined antigen as a mixed T cell population or as a purified Tcell subset. For example, purified CD8⁺ T cells can be co-cultured withantigen-exposed dendritic cells to elicit an antigen-specific CTL. Incertain embodiments, early elimination of CD4⁺-T cells can prevent theovergrowth of CD4⁺ cells in a mixed culture of both CD8⁺ and CD4⁺ Tcells. Alternatively, mixed populations of CD4⁺ and CD8⁺ T cells can beco-cultured with dendritic cells to elicit a response specific to anantigen encompassing both a cytotoxic and T_(H) immune response.

Such stimulated T cells optionally can be reinfused into a subjects.(See, e.g., Riddle and Greenberg, J. Antimicrobial Chemotherapy45:35-43, 2000; Correale et al., J. Neuroimmunology 107:130-39, 2000;the disclosures of which are incorporated by reference herein.) Forexample, immature dendritic cells can be contacted with antigen (e.g.,PSMA) and matured to form mature dendritic cells. T cell can be isolatedfrom a subject, contacted with the mature dendritic cells ex vivo, andthen re-administered to the subject. For example, doses of about 1×10⁷to about 5×10⁹ CD8⁺ T cells can be administered to a subject weekly, orbi-weekly, for a period of 1-4 months, or more. Alternatively, themature dendritic cells can be administered directly to the subject.Typically, about 1×10⁷ dendritic cells are used per administration to apatient.

Typically a leukaphoresis product from a donor treated with a stem cellmobilization agent comprises about 1 to 5% cells, about 5 to about 20%granulocytes, about 40 to about 60% lymphocytes around about 10 to about25% monocytes with significant amounts of red blood cells and platelets.Using a TFF device of the present invention with a filter having a poresize of about 3 to about 5.5 microns results in an enriched leukocytepopulation comprising about 60 to about 70% monocytes, almost nogranulocytes, about 10% lymphocytes and about 10 to about 40% CD 34⁺cells. This enriched leukocytes population can be used as set forthbelow.

In other embodiments, the methods of the present invention are used toobtain a cell subset other than monocytes or monocytic dendritic cellprecursors. For example, an enriched population of leukocytes can beused as a source of hematopoietic stem cells for, e.g., allogeneic orautologous transplantation. In particular embodiments, the enrichedpopulation of leukocytes is further enriched for the stem cellsfollowing the tangential flow separation procedure. Methods forenrichment of hematopoietic stem cells from a source of peripheral bloodleukocytes are known in the art and can be adapted for use with anenriched population of leukocytes isolated as described herein. Forexample, an enriched population of leukocytes can be further enrichedfor CD34⁺ cells using, e.g., immunomagnetic separation techniques (see,e.g., Rowley et al., Bone Marrow Transplant. 21:1253, 1998;Denning-Kendall et al., Br. J. Haematol. 105:780, 1999). In addition, tofurther increase stem cell yields, a cell population enriched inmonocytes subsequent to TFF as described in the present invention can becultured in the presence of, for example, about 50 ng/ml M-CSF in mediumcontaining fetal serum to derive CD34⁺ cells. The culture must becarried out in a non-adhesive cell culture container such as a TEFLON®culture bag. Further, peripheral blood donors can be subjected to a stemcell mobilizing regimen prior to collection of peripheral blood andseparation of leukocytes by TFF. Various mobilizing agents forincreasing efficiency of stem cell harvest are known in the art. Forexample, donors can be treated with GM-CSF, G-CSF, AMD3100 (or otheragent that inhibits CXCR-4 function), and/or mobilizing chemotherapeuticagents such as, e.g., high- or low-dose cyclophosphamide (see, e.g.,Deliliers et al., Leuk. Lymphoma, 43:1957, 2002). The blood donor can bethe patient to receive the transplant, a close relative, a HLA-matchedindividual, or the like.

In yet another embodiment, the methods of the present invention are alsoused to obtain a non-stem cell subset such as, for example, a cellpopulation enriched in progenitor cells (e.g., hematopoietic orendothelial progenitor cells) or cells that secrete a factor of interest(e.g., hematopoietic or angiogenic growth factors). For example,circulating endothelial progenitor cells (CEPs) can be identified as asubset of circulating CD34⁺ cells by, e.g., coexpression of VEGFR-2 andAC133 (as well as, e.g., VE-cadherin and E-selectin). (See, e.g.,Peichev et al., Blood 95:952, 2000.) An enriched population ofleukocytes can be further enriched for CEPs using, for example,immunomagnetic separation techniques with antibodies directed to VEGFR-2and AC133. Also, CEPs can be mobilized prior to TFF by treatment withcytokines such as, e.g., VEGF. (See, e.g., Gill et al., Circ Res.,88:167, 2001). Further, in yet other embodiments, endothelial-likecirculating angiogenic cells (CACs) (which secrete, e.g., VEGF, HGF,G-CSF, and GM-CSF) are obtained by culturing an enriched population ofleukocytes with, e.g., VEGF, bFGF, IGF-1, EGF, and FBS on afibronectin-coated surface and then discarding non-adherent cells (see,e.g., Rehman et al., Circulation 107:1164, 2003).

In addition, the enriched population of leukocytes can be cultured toinduce expansion of pluripotent progenitor or stem cells. For example,CD34⁺ stem cells can be expanded by culture with hematopoietic growthfactors such as, e.g., a combination IL-1, IL-3, IL-6, stem cell factor(SCF), granulocyte-monocyte colony-stimulating factor (GM-CSF) and G-CSF(see, e.g., Sun et al., Haematologica 88:561, 2003). Alternatively, forexample, a population enriched for monocytes can be treated with, e.g.,M-CSF, LIF, and/or IL-6 to obtain pluripotent “f-macrophages” (f-MΦ),which morphologically resemble fibroblasts and, unlike standardmacrophages, display elevated levels of CD34 (See Zhao et al., Proc.Natl. Acad. Sci. USA 100:2426, 2003.) The progenitor or stem cells cansubsequently be treated with any of various cytokines and growth factorsto induce differentiation into hematopoietic or non-hematopoieticlineages.

In other embodiments, an enriched population of leukocytes can becultured under conditions suitable for inducing differentiation (e.g.,differentiation of progenitor cells or transdifferentiation of moredifferentiated cells types such as, for example, monocytes ormonocyte-derived dendritic cells). (As used herein,“transdifferentiation” refers to a processs of phenotypic modulation ofa differentiated cell, generally without the need for any cell division,whereby the differentiated cell differentiates into a morphologicallyand/or functionally different cell type.) For example, in addition todifferentiation into dendritic cells, monocytes can be transformed intoother hematopoietic or non-hematopoietic cell types, including, e.g.,macrophages, osteoclasts, and endothelial-like cells, depending onculture conditions (see, e.g., Becker et al., J. Immunol. 139:3703,1987; Nicholson et al., Clin Sci. 99:133, 2000; Havemann et al., inNovel Angiogenic Mechanisms: Role of Circulating Progenitor EndothelialCells 47-57 (Nicanor I. Moldovan eds., 2003)). In one embodiment, anenriched population of monocytes or monocyte-derived dendritic cells istransdifferentiated into endothelial-like cells by culture with, e.g.,VEGF, bFGF, IGF-1, hydrocortisone, and FCS on a fibronectin-coatedsurface. (See Havemann et al., supra.) Also, an enriched population ofleukocytes can be cultured under conditions that induce differentiationof relatively undifferentiated cell subsets (e.g., pluripotentprogenitor and stem cells) into hematopoietic or non-hematopoieticlineages using any of various cytokines or growth factors. For example,monocyte-derived pluripotent stem cells (f-MΦ) can be induced todifferentiate into standard macrophages, T lymphocytes, epithelialcells, neuronal cells, endothelial cells, or hepatocytes by treatmentwith, e.g., LPS, IL-2, EGF, NGF, VEGF, or HGF, respectively. (See Zhaoet al., supra) Such differentiation can be induced prior to or followingcell expansion such as, for example, described supra.

The following examples are provided merely as illustrative of variousaspects of the invention and should not be construed to limit theinvention in any way.

EXAMPLE 1 TFF Device with Remover Unit and Recovery Unit in LoopConfiguration

One embodiment of the present invention comprises a configuration havinga remover unit (1) and a recovery unit (2) in a loop configuration ofthe tangential flow filtration device. (FIG. 1A) The remover unitincluded a housing having two chambers (a cross-flow chamber (3) and afiltrate chamber (4)), separated by a microporous filter (5) (142 mm indiameter) having a pore size of about 1 micron to about 10 microns. Thecross-flow chamber included a fluid inlet (6) and a fluid outlet (7).The filtrate chamber included a filtrate outlet (8). The recovery unitincluded a housing (9) containing a return inlet (10), a return outlet(11), a sample inlet (12), and a solution inlet (13). In certainembodiments the sample inlet and solution inlet are the same, but can beseparate. Sample (e.g. blood, blood preparations, or a preparedpopulation of leukocytes) was introduced into the recovery unit by thesample inlet (12), and was withdrawn through the return outlet (11) tothe remover unit by action of a recirculating pump (14). Sample wasintroduced into the remover unit through the fluid inlet (6) and flowedacross the microporous membrane (5), such that the fluid's movement wasdirected at a tangent to the direction of filtration. The fluid inlet(6) was positioned generally perpendicular to the radius of the filter.The relative inlet, filtration and outlet rates induced a vortex, thecenter of which drew components in the blood preparation not passingthrough the filter, to the fluid outlet (7) and back to the recoveryunit (2). The flow of fluid between the recovery unit and the removerunit was controlled by the recirculation pump (14). The removal offiltrate from the remover unit was controlled by a filtrate pump (15).

EXAMPLE 2 Retention of Leukocytes Following TFF—Effects of RecirculationRate and Filtrate Rate on Retention Efficiency

In this example the enrichment of leukocytes using a TFF device asdescribed in Example 1, which accommodated polyester membranes of 142 mmin diameter, was demonstrated. A sample of leukopheresis product wassubjected to TFF in a device under various conditions, and the selectiveretention of leukocytes was assessed. In one set of studies, TFF wasperformed for 10 ml of leukopheresis product using a 3 micron filter,with a filtration rate of 15 ml/min (for 17 min) and at variousrecirculation (input) rates (e.g., 1680, 1380, 1080, 870, and 540ml/min). Most of the leukocytes were generally retained in the retentate(i.e., less than about 10 percent of the leukocytes in the filtrate)unless the recirculation rate was lower than 1080 ml/min (FIG. 2; note:the corresponding actual recirculation rates designated Max, 10, 9, 8,7, and 6 were 1680, 1380, 1080, 870, and 540 ml/min, respectively).

In another set of studies, TFF was performed for 10 ml of leukopheresisproduct in the TFF device of Example 1 using a 3 micron filter, with arecirculation (input) rate of 1080 ml/min at three different filtrationrates (11, 15, and 19.6 ml/min). About 250 ml of filtrate was collectedper study. Neither the 19.6 ml/min nor the 11 ml/min filtration rate wassubstantially more beneficial than the 15 ml/min filtration rate forretention of leukocytes (FIG. 3).

EXAMPLE 3 Selective Removal of Erythrocytes From Leukopheresis Product

In this example the selective removal of erythrocytes and the effects ofrecirculation rate, filtration rate and sample concentration on theseparation efficiency using the TFF device of Example 1 wasdemonstrated. Samples of leukopheresis product were subjected to TFFunder the conditions as established in Example 2, and the selectiveremoval of erythrocytes was assessed. As in Example 2, TFF was performedfor 10 ml of leukopheresis product using a 3 micron filter, with afiltration rate of 15 ml/min (for 17 min) and at various recirculation(input) rates (e.g., 1680, 1380, 1080, 870, and 540 ml/min). It wasdetermined that generally, reducing the recirculation rate promoted moreerythrocytes to pass into the filtrate (FIG. 4; note: the correspondingactual recirculation rates for Max, 10, 9, 8, 7, and 6 were 1680, 1380,1080, 870, and 540 ml/min, respectively). It can be seem that whiledecreasing the recirculation rate did increase the removal oferythrocytes from the retentate, at a recirculation rate lower than 1080ml/min the yield of leukocytes in the retentate was reduced (FIG. 4).

As in Example 2, TFF was performed for 10 ml of leukopheresis product inthe same TFF device using a 3 micron filter, with a recirculation(input) rate of 1080 ml/min at three different filtration rates (11, 15,and 19.6 ml/min). About 250 ml of filtrate was collected per study.Neither the 19.6 ml/min nor the 11 ml/min filtration rate wassubstantially more beneficial than the 15 ml/min filtration rate for theselective removal of erythrocytes (FIG. 5). The filtration rate of 19.6ml/min reduced the erythrocytes in the retentate more than 15 ml/min,but as seen in Example 2 the 15 ml/min filtration rate resulted in agreater yield of leukocytes in the retentate (FIG. 3).

Referring to FIG. 6, the effect of increasing the concentration ofleukopheresis material in the sample was also studied. Fifty ml (50 ml)of leukopheresis material, diluted 1:5 in PBS+heparin+DNase I, wassubjected to TFF using a device with a filter having a 3 micron poresize. The percentage of erythrocytes (designated “RBC”) in the retentatewas about the same, about 100% of input, if the filtration rate wasabout 15 ml/min or 19.6 ml/min. However, if the 50 ml of leukopheresisproduct was diluted 1:2 in the same buffer, only 40% of the inputerythrocytes were retained in the retentate, showing that loading ofhigher sample concentrations can also promote the separation oferythrocytes from leukocytes (designated “WBC”).

EXAMPLE 4 Selective Removal of Erythrocytes from LeukopheresisProduct—Effect of Pore Size on Removal of Erythrocytes

To confirm that the embodiment of the TFF device set forth in Example 1would perform similarly upon scale-up, TFF of 120 ml of leukopheresisproduct (½ of an entire unit) was compared in devices having filterswith a pore size of 3 micron verses 4.5 micron. The recirculation(input) rate was 1680 ml/min, and the filtration rate was 15 ml/min.Referring to FIG. 7, for TFF performed using a 4.5 micron filter, about80% of the erythrocytes (designated “RBC”; dark shaded bars) wereremoved from the retentate, while about 62% of input leukocytes(designated “WBC”; light hatched bars), or greater than about 70% ofinput monocytes, were retained. In contrast, using the 3 micron filter,about 65% of input leukocytes were retained in the retentate, but only3% of the erythrocytes were removed.

Referring to FIG. 8, TFF of 45 ml of leukopheresis product was comparedin devices having filters with a pore size of 4.5 micron versus 8micron. The recirculation (input) rate was 1680 ml/min, the filtrationrate was 15 ml/min, and the time was 60 min. For TFF performed using a4.5 micron filter, about 99% of the erythrocytes (designated “RBC”; darkshaded bars) were removed from the retentate, while about 90% of inputleukocytes (designated “WBC”; light hatched bars) were retained. For the8 micron filter, about 98% of input erythrocytes were removed, but only4% of the leukocytes were retained, showing that using a 4.5 micronfilter promoted equal removal of erythrocytes to the larger 8 micronpore size filter, but a better retention of leukocytes (designated“WBC”) was obtained.

EXAMPLE 5 Selective Enrichment of Leukocytes with Removal of Plateletsand Plasma

In this example samples of leukopheresis product were subjected to TFFin the device described in Example 1 under various conditions, and theselective removal of platelets and plasma was assessed. Referring toTable 1, experiments 1 to 6, 45 ml of leukopheresis product wassubjected to TFF with a filter having either a 4.5 micron or a 8 micronpore size, the recirculation rate (input rate) was either 1690 or 1880ml/min, and the filtration rate was 15 ml/min. The filtration wascarried out for either 60 or 90 min as indicated. Following TFF, theretentate was assayed for leukocytes, platelets, and plasma. In allexperiments, between 92 to about 100% of the input platelets and about97 to about 99% of the input plasma were removed from the retentate,independent of pore size (4.5 micron or 8 micron), recirculation (input)rate (1690 or 1880 ml/min), and time (60 min or 90 min). For experiment7, an entire leukopheresis product (250 ml) was subjected to TFF using a4.5 micron pore size, 1690 ml/min recirculation (input) rate, 15 ml/minfiltration rate, for a 90 min duration. Following TFF, the retentate wasassayed and found to have about 84% of the input leukocytes, but only 2%of the input platelets and 3% of the input plasma. TABLE 1 Effect ofFilter Pore Sizes on Retention of PBMC % Input % Input % InputLeukoperesis Pore Recirculation WBC in Platelets in Plasma in Input SizeRate Time Retentate Retentate Retentate Exp 1 45 ml 4.5 μm 1690 ml/min60 min 88 2.0 2.0 Exp 2 45 ml 4.5 μm 1690 ml/min 60 min 67 8.0 2.0 Exp 345 ml 4.5 μm 1690 ml/min 60 min 79 3.0 1.0 Exp 4 45 ml 4.5 μm 1888ml/min 60 min 92 0.0 1.0 45 ml   8 μm 1888 ml/min 60 min 4 0.0 1.0 Exp 545 ml 4.5 μm 1690 ml/min 60 min 130 3.0 3.0 45 ml 4.5 μm 1880 ml/min 60min 47 3.0 2.0 Exp 6 45 ml 4.5 μm 1690 ml/min 60 min 80 1.0 2.0 45 ml4.5 μm 1690 ml/min 90 min 72 0.2 2.0 Exp 7 250 ml  4.5 μm 1690 ml/min 90min 84 2.0 3.0

These experiments show that TFF removes most of the platelets and plasmafor multiple pore sizes (4.5 micron or 8 micron), recirculation (input)rates (1690 or 1880 ml/min), volumes of leukopheresis sample (45 ml to250 ml), and time (60 min to 90 min).

EXAMPLE 6 Selective Enrichment of Leukocytes from Whole LeukopheresisProducts

To confirm that this embodiment of the TFF device would performreproducibly upon scale-up to larger sample sizes, a TFF devicedesignated a 5× device was used with 250 ml of leukopheresis product (anentire unit) using a 4.5 micron pore size filter, a recirculation rateof 1680 ml/min, and a filtration rate of 15 ml/min and compared with thelower input volume. Referring to FIG. 9, TFF was performed for 90 min onthree different leukopheresis products on different days. Between 80 to95% of the erythrocytes were removed from the retentate, while about 80to about 100% of input monocytes were retained. Following TFF for Exp 1in FIG. 9, the retentate was also assayed and found to have about only2% of the input platelets and 3% of the input plasma (designated Exp 7in Table 1). These data demonstrated that TFF can reproducibly enrichfor leukocytes and preferentially remove erythrocytes, platelets, andplasma from the retentate.

EXAMPLE 7 TFF Device for Selectively Enriching A Blood Preparation forMonocytes

In addition to enrichment for leukocytes, selective enrichment ofmonocytes from blood preparations was tested using a TFF apparatus. Thisparticular embodiment of the device comprised an inverted (“type IV”)configuration designed to change the flow dynamics by altering thedirection of gravitational force across the membrane (FIG. 1C). Twohundred thirty-five (235) ml of leukopheresis product was subjected toTFF for 90 minutes using a 4.5 micron pore size filter, a recirculation(input) rate of 1680 ml/min, and a filtration rate of 15 ml/min. After60 minutes of TFF, in order to increase the effective filtrate, the voidvolume was reduced to about 120 ml. The input, retentate, and filtratewere each assayed for cell content. Whereas the input had 32% monocytesand 65% lymphocytes, the retentate was found to have about 71% monocytescompared to 22% lymphocytes. The filtrate contained 1.5% monocytescompared to 83% lymphocytes. (See Table 2). TABLE 2 Enrichment ofMonocytes Using Type IV (Inverted) TFF Configuration Input RetentateFiltrate Monocyte No.(×10⁹) 2.85 1.37 0.861 Lymphocyte No. (×10⁹) 5.670.428 4.86 Granulocyte No. (×10⁹) 0.133 0.0571 0.0797 RBC No. (×10⁹)64.6 1.37 78.9 WBC No. (×10⁹) 8.78 1.93 5.86 % Monocytes 32.46 71.1 14.7% Lymphocytes 64.54 22.17 82.88 % Granulocytes 0.0151 2.96 1.36 Unlysed(×10⁹) 73.4 3.30 84.8

EXAMPLE 8 Generation of Dendritic Cells From Cells Isolated by TFF

In this example the cell population that was isolated and purified byTFF was cultured in standard conditions for the maturation of dendriticcells from monocytic dendritic precursor cells. The population of cellsisolated by TFF contained approximately 58.6% monocytes, 22% lymphocytesand 12.5% granulocytes. This mixture of cells was introduced into atissue culture bag in X-Vivo15 media and 500 U each of IL-4 and GM-CSF.After five days, approximately 50% of the cells were harvested, stainedfor DC markers and analyzed by flow cytometry. The other approximately50% of the cells were exposed to maturation agents, BCG (2.8×10⁵ pfu/ml)and IFNγ (1000 U/ml). After 24 h those cells were also harvested andanalyzed in the same manner. Table 3 shows the results of theseanalyses. The values provided represent the percentage of positive cellsamong the dendritic cells, i.e., after gating on large cells. TABLE 3Detection of Cell Surface Markers on Immature and Mature DendriticCells. Marker Immature DC (% positive) Mature DC (% positive) CD14 10 2CD11c 99 96 CD1a 70 69 CD80 75 74 CD83 7 34 CD86 90 89 CD54¹ 100 98 MHCII¹ 56 72¹These markers show a significant increase in mean fluorescenceintensity (MFI) among positive cells after maturation

Based on the forward light scatter and side scatter parameters of theflow-cytometric analysis it was determined that the immature dendriticcell population contained approximately 71% live DC, 21% lymphocytes and7% other cells (mostly dead cells of unknown origin). The maturedendritic cell population contained 61% live DC, 21% lymphocytes and 15%other cells. These results demonstrated that monocytes purified directlyfrom leukopheresis material by TFF can be efficiently converted into DCusing standard culture conditions maturing dendritic cells.

EXAMPLE 8 Isolation of Monocytes on Glass Beads

An enriched population of leukocytes is isolated by TFF according to anyof the previous examples. During TFF, the buffer is replaced with AIM-Vmedia (Gibco-Life Science) containing 1% heat-inactivated autologousplasma (binding media). Glass beads (20 grams) are prepared by washingtwice in binding media and are subsequently placed in a 60 millilitersyringe fitted with a frit to retain the beads to form a column bed.(Alternatively, Plastic Plus microcarrier beads (treated styrenecopolymer beads from SoloHill Engineering, Inc.) or HilleX microcarrierbeads (styrene copolymer beads from SoloHill Engineering, Inc.) can beused. The binding media is then drained from the column bed by gravityflow. The enriched population of leukocytes from TFF is applied to thecolumn, and any flowthrough is collected. Binding media is added toprovide a small layer of liquid above the column bed. The column withcells is incubated at 37° C. for 30 minutes.

After incubation, the column port is opened, and the flowthrough iscollected. The column bed is then washed six times with binding media(35 ml/wash) administered and removed multiple times through the columnport to allow gentle resuspension of the beads. These washes arefollowed by two washes with phosphate buffered saline (“PBS”). Cellcounts are obtained for all washes and the original flow through, andthey are analyzed by forward and side scatter FACS analysis to determinethe percentage of monocytes present. After completing the washes, thebound monocytes are eluted from the beads using PBS/0, 4% EDTA (w/v),followed by one more PBS wash. The cells that are obtained in thesefractions are analyzed in the same manner as the washes. The fractionsrich in monocytes are pooled.

EXAMPLE 9 Differentiation of Dendritic Cells From Monocytes Eluted fromGlass Beads

Monocytes are washed two times with 30 ml of PBS, and resuspended inculture media (X-VIVO 15 (Biowhittaker Corp.) with 500 U GM-CSF/ml and500 U Interleukin 4/ml). A portion (⅔) of the cell suspension is thentransferred to a rotary bioreactor (Synthecon) and cultured for 6 daysat 37° C. in a humidified environment containing 5% CO₂. Afterculturing, the cell population comprises about 70% immature dendriticcells, based on cell size, granularity and cell surface markers.

EXAMPLE 10 Activation of Immature Dendritic Cells with Prostate SpecificAntigen

Monocytes are isolated from a prostate cancer patient as described inthe previous examples. The monocytes are cultured in tissue culture bagsin X-VIVO 15 tissue culture media supplemented with GM-CSF andInterleukin 4 (500 U/ml each) for 6 days at 37° C. The resultingimmature dendritic cells are then exposed to prostate specific antigen(PSMA) (isolated as described in U.S. Pat. No. 5,788,963) added to theculture media. The immature dendritic cells are then differentiated toform mature dendritic cells using a maturation agent. The mature(activated) dendritic cells are added to a T cell proliferation assay. Tcell cultures are incubated in a humidified 37° C. incubatorsupplemented with 5% CO₂ for 5 days prior to addition of 1 μCi³H-Thymidine/well of a microtiter plate. After a 24 hour incubation, thecells are harvested in a semi-automatic cell harvester (Skatron,Stevina, Va.), and the radioactivity of the collected cells isdetermined. T cell proliferation is assessed by measurement of average³H-TdR incorporation.

EXAMPLE 11 Capacity of Cultured Mature Dendritic Cells to PresentAntigen

To assess the capacity of the cultured, mature dendritic cells topresent antigen to and stimulate autologous T cells from the samepatients, T cell proliferation assays are conducted as described above(Example 10). Tetanus toxoid is chosen as the representative antigen inthese experiments to determine whether patients' memory T cells can beactivated in vitro. Autologous T cells cultured with the patient'sdendritic cells and Tetanus toxoid will proliferate at levelssignificantly higher than background levels (in the absence dendriticcells) and at levels significantly higher than T cells cultured withmature (activated) dendritic cells without Tetanus toxoid (i.e., showingan autologous mixed lymphocyte reaction). Thus, the presentation ofTetanus toxoid by dendritic cells is useful for T cell proliferation.

EXAMPLE 12 Stimulation of Autologous T Cells

Mature (activated) dendritic cells specific for prostate cancer are usedto stimulate autologous T cells of a prostate cancer patient. A crudecellular lysate of LNCaP cells, a metastatic prostate cancer cell line,is used as a representative prostate cancer antigen in a T cellproliferation assay generally as described in U.S. Pat. No. 5,788,963(the disclosure of which is incorporated by reference herein). Asignificant increase in ³HTdR incorporation is observed when both matureactivated dendritic cells and LNCaP lysates are included in the T cellcultures.

EXAMPLE 13 Administration of Stimulated Autologous T Cells to a Subject

T cells are prepared by leukopheresis from a subject. The T cells arecontacted with mature activated dendritic cells. The dendritic cells arematured after contacting with a crude cellular lysate of LNCaP cells, ametastatic prostate cancer cell line. Following contacting, the T cellsand dendritic cells are cultured and expanded. The expanded, activated Tcells are administered to the subject at a dose of about 10⁷ to about5×10⁹ cells per dose.

EXAMPLE 14 Conversion of Monocytes to CD34⁺ Stem Cells

The percentage of CD34⁺ cells in peripheral blood is extremely low,ranging from about 0.01% to about 0.1%. CD34⁺ cells are recognized asthe cell type necessary for successful transplantation of hematopoieticfunction. Monocytes can be isolated from peripheral blood using thedevice and methods described above, yielding 1 to 2×10⁹ monocytes. Thesecells can then be cultured in 50 ng/ml M-CSF in medium containing fetalbovine serum to derive CD34⁺ cells. The cultures must be performed in anon-adhesive environment such as a TEFLON® culture bags. Cultures instandard polystyrene tissue culture flasks do not develop into CD34⁺cells. Analysis of the large cells in the culture revealed that theCD34⁺ cell were found in the larger size range. TABLE 4 Expression ofCD34 on Monocytes after culture in M-CSF Percentage of CD34⁺ CellsCulture in culture bag 21 Gating on large cells 58 Culture in flask 6Gating on large cells 11CD34⁺ cells prepared by this method can be used transplantation toreconstitute the recipient's bone marrow or may be further cultured inthe presence of other cytokines to generate endothelial cells for use intreating, eg. myocardial infarction, etc. and the like.

The examples are provided herein are intended to illustrate but not tolimit the scope of the claimed invention. Other variants of theinvention will be readily apparent to those of ordinary skill in the artand encompassed by the appended claims. All publications, patents,patent applications and other references cited herein and are alsoincorporated by reference herein.

1. A tangential flow filtration device for preparing a cell populationenriched for leukocytes, comprising: a remover unit having a cross-flowchamber, a filtrate chamber and a filter disposed therebetween, thefilter in fluid communication with the cross-flow chamber and thefiltrate chamber; the cross-flow chamber having an inlet and an outlet,the inlet disposed to introduce a sample of blood constituentscomprising leukocytes into the cross-flow chamber and parallel to thesurface of the filter; and the outlet centrally disposed in a portion ofthe cross-flow chamber opposite the filter surface; the filter having anaverage pore size ranging from about 1 to about 10 microns; such thatflow of the sample across the filter enriches the sample of bloodconstituents for leukocytes.
 2. The device according to claim 1, furthercomprising: a means for providing a predetermined input rate of thesample to the inlet of the cross-flow chamber; a means for controlling afiltration rate of filtrate through the filter and into the filtratechamber; and wherein the filtration rate controlling means limits thefiltration rate to less than the unopposed filtration rate for thefilter.
 3. The device according to claim 1 or claim 2, wherein thefilter pore size is about 3 microns to about 7 microns.
 4. The deviceaccording to claim 1 or claim 2, wherein the filter pore size is about 3microns to about 5.5 microns.
 5. The device according to claim 1 orclaim 2, further comprising: a source of blood constituents in fluidcommunication with the cross-flow chamber inlet.
 6. The device accordingto claim 5, wherein the source of blood constituents is a leukopheresisdevice.
 7. The device according to claim 1 or claim 2, furthercomprising: a recovery unit comprising an inlet and an outlet, thecross-flow chamber and the recovery unit interconnected in loop format,wherein the cross-flow chamber inlet is in fluid communication with therecovery unit outlet, and the cross-flow chamber outlet is in fluidcommunication with the recovery unit inlet.
 8. The device according toclaim 7, wherein the recovery unit further comprises a sample inlet anda wash inlet.
 9. The device according to claim 8, further comprising asource of replacement liquid in fluid communication with the wash inlet.10. The device according to claim 9, wherein the replacement liquid isan isotonic buffer or tissue culture media.
 11. The device according toclaim 1 or claim 2, wherein the cross-flow chamber is cylindrical andthe outlet is located opposite the center of the filter andperpendicular to a surface of the filter.
 12. The device according toclaim 1 or claim 2, further comprising a cell-processing apparatus influid communication with the remover unit.
 13. The device according toclaim 12, wherein the cell processing apparatus comprises beads.
 14. Thedevice according to claim 12, wherein the cell processing apparatuscomprises a means for culturing the cell population enriched forleukocytes.
 15. The device according to claim 14, wherein the means forculturing comprises: a vessel having a first port and a second port; amonocytic dendritic cell precursor adhering substrate, the substrate influid communication with the first port and the second port; a screenfor retaining the substrate within the vessel, the screen having a poresize sufficient to allow passage of monocytic dendritic cell precursorsand dendritic cells therethrough; a drain line in fluid communicationwith the first port; and a collection line in fluid communication withthe first port.
 16. The device according to claim 15, further comprisinga plurality of fluid sources in fluid communication with the first portor the second port.
 17. The device according to claim 15, furthercomprising a sealable tissue culture vessel adapted to asepticallyreceive the monocytic dendritic cell precursors.
 18. The deviceaccording to claim 17, wherein the sealable tissue culture vessel is atissue culture bag, flask or bioreactor.
 19. The device according toclaim 15, wherein the fluid sources comprise binding media, washingbuffer and elution buffer.
 20. The device according to claim 15, furthercomprising a pump in fluid communication with the plurality of fluidsources and the first port.
 21. The device according to claim 15,further comprising: a temperature control means to maintain thesubstrate at a predetermined temperature.
 22. The device according toclaim 21, wherein the temperature controlling means is a heater.
 23. Atangential flow device for enriching a sample of blood constituents forleukocytes, comprising: a remover unit comprising a cross-flow chamber(3) and a filtrate chamber (4) separated by a filter (5), wherein thecross-flow chamber (3) has an inlet (6) and an outlet (7), the outletcentrally disposed in an upper portion of the chamber, and wherein theinlet is disposed above the filter and introduces fluid into thecross-flow chamber substantially parallel to the filter; a means forproviding a predetermined input rate (14) of the sample through thecross-flow chamber inlet; and a means for reducing a filtration rate(15) through the filter; wherein the filter has a pore size of about 3microns to about 7 microns; and whereby the sample is enriched forleukocytes in a retentate in the cross-flow chamber.
 24. A tangentialflow device for enriching a sample comprising blood constituents forleukocytes, comprising: a remover unit (1) having a cross-flow chamber(3) and a filtrate chamber (4) separated by a filter (5), the cross-flowchamber having an inlet (6) and an outlet (7), the outlet disposed abovethe inlet and centrally disposed in an upper portion of the chamber, andwherein the filter is disposed below and substantially parallel to thecross-flow chamber inlet; means for providing a predetermined input rate(14) of the sample through the cross-flow chamber inlet; means forproviding a predetermined filtration rate (15) of the fluid through thefilter, wherein the predetermined filtration rate is about one-fifth toabout one one-hundredth of the predetermined input rate; and means forproviding a predetermined concentration of blood cells in the sample,wherein the predetermined concentration of blood cells is about 10⁷ toabout 10¹⁰ cells per milliliter; wherein the filter has pores having apore size of about 3 microns to about 7 microns; and whereby the sampleis enriched for leukocytes in a retentate in the cross-flow chamber. 25.A method for separating leukocytes from a sample of blood constituentsfrom a subject wherein the sample comprises leukocytes, the methodcomprising: (1) introducing the sample into a remover unit through aninlet in the remover unit; (2) subjecting the sample to cross-flowsubstantially parallel to a filter having a pore size of about 1 toabout 10 microns; (3) subjecting the fluid to filtration through thefilter; and (4) selectively removing non-leukocyte blood constituentsfrom the sample to form a cell population enriched for leukocytes. 26.The method according to claim 25, further comprising: preparing thesample from the subject by leukopheresis, density centrifugation,differential lysis, filtration, or preparation of a buffy coat, forintroduction in the remover unit.
 27. The method according to claim 25,wherein the non-leukocyte blood constituents include plasma andplatelets.
 28. The method according to claim 25, wherein thenon-leukocyte blood constituents include erythrocytes.
 29. The methodaccording to claim 25, wherein the leukocytes comprise monocytes. 30.The method according to claim 25, further comprising repeating steps(1), (2), and (3) at least two times to form cell population enrichedfor leukocytes.
 31. The method according to claim 25, wherein theenriched cell population comprises at least about 20% leukocytes. 32.The method according to claim 25, wherein the enriched cell populationcomprises at least about 60% leukocytes.
 33. The method according toclaim 25, further comprising inducing a vortex motion of the sample inthe cross-flow chamber.
 34. The method according to claim 25, furthercomprising washing the cell population enriched for leukocytes with awash solution.
 35. The method according to claim 25, further comprisingpreparing monocytic dendritic cell precursors from the cell populationenriched for leukocytes.
 36. The method according to claim 35, whereinthe isolation of monocytic dendritic cell precursors comprises:contacting a monocytic dendritic cell precursor adhering substrate withthe cell population enriched for leukocytes; allowing monocyticdendritic cell precursors in the cell population to reversibly adhere tothe substrate to form complexes comprising monocytic dendritic cellprecursors and substrate; separating the complexes from the non-adheringleukocytes to obtain complexes comprising monocytic dendritic cellprecursors; and culturing the monocytic dendritic cell precursors todifferentiate the precursors to form immature or mature dendritic cells.37. The method according to claim 36, wherein the monocytic dendriticcell precursors are eluted from the substrate prior to culturing. 38.The method according to claim 36, wherein the monocytic dendritic cellprecursors are cultured on the substrate.
 39. The method according toclaim 36, wherein the substrate comprises glass, polystyrene, plastic orglass-coated polystyrene microbeads.
 40. A method for enriching a sampleof blood constituents for leukocytes, comprising: (1) introducing thesample into a tangential flow filtration (TFF) unit, the TFF unitcomprising a cross-flow chamber, a filtrate chamber, and a filter influid communication with the cross-flow chamber and the filtratechamber, the filter having a pore size of about 1 to about 10 microns;(2) recirculating the sample through the TFF unit at a predeterminedinput rate and a predetermined filtration rate, the predetermined inputrate at least five times the predetermined filtration rate; wherein thepredetermined filtration rate is less than the unopposed filtration ratefor the filter; and (3) isolating a cell population enriched forleukocytes.
 41. The method according to claim 40, wherein the enrichedcell population is substantially free of non-leukocyte bloodconstituents.
 42. The method according to claim 40, further comprising:collecting blood from a subject and preparing the sample from the bloodby leukopheresis, density centrifugation, differential lysis,filtration, or preparation of a buffy coat.
 43. The method according toclaim 40, wherein the non-leukocyte blood constituents include plasmaand platelets.
 44. The method according to claim 41, wherein thenon-leukocyte blood constituents include erythrocytes.
 45. The methodaccording to claim 40, wherein the leukocytes comprise monocytes. 46.The method according to claim 40, wherein the enriched cell populationcomprises at least about 20% leukocytes.
 47. The method according toclaim 40, wherein the enriched cell population comprises at least about60% leukocytes.
 48. The method according to claim 40, wherein the sampleflows in a vortex motion in the cross-flow chamber.
 49. The methodaccording to claim 40, further comprising washing the enriched cellpopulation with a wash solution.
 50. The method according to claim 40,further comprising preparing dendritic cells from the enriched cellpopulation.
 51. The method according to claim 50, wherein the dendriticcells are prepared by: contacting a monocytic dendritic cell precursoradhering substrate with the enriched cell population; allowing monocyticdendritic cell precursors in the enriched cell population to reversiblyadhere to the substrate to form complexes comprising monocytic dendriticcell precursors and substrate; separating the complexes from thenon-adhering leukocytes to obtain complexes comprising monocyticdendritic cell precursors; and culturing the monocytic dendritic cellprecursors to differentiate the precursors to form immature or maturedendritic cells.
 52. The method according to claim 51, wherein thesubstrate comprises glass, polystyrene, plastic or glass-coatedpolystyrene microbeads.
 53. The method according to claim 51, furthercomprising isolating the immature or mature dendritic cells.
 54. Themethod according to claim 45, wherein the monocytes are cultured withcytolines that promote the differentiation of monocytes into dendriticcells.
 55. The method according to claim 54, wherein the cytokines areGM-CSF, GM-CSF and IL-4.
 56. The method according to claim 54, whereinthe dendritic cells are matured to mature dendritic cells.
 57. Themethod according to claim 54, wherein the dendritic cells are culturedwith an antigen under conditions conducive for processing the antigen toform antigen loaded dendritic cells.
 58. The method according to claim57, further comprising the step of administering the antigen loadeddendritic cells to an individual.
 59. The method according to claim 57,wherein the antigen loaded dendritic cells are cultured with amaturation agent to mature the cells into mature antigen presentingdendritic cells.
 60. The method according to claim 40, wherein thefilter has a pore size of about 3 to about 5.5 microns.
 61. The methodaccording to claim 60, wherein the leukocytes comprise CD34⁺ cells. 62.The method according to claim 61, wherein the sample of bloodconstituents is from a donor tat has been treated with at least one stemcell mobilizing agent.
 63. The method according to claim 62, wherein thestem cell mobilizing agent is G-CSF or cyclophosphamide.
 64. The methodaccording to claim 61, further comprising enriching the leukocytes forthe CD34⁺ cells.
 65. The method according to claim 64, wherein theenrichment of leukocytes for the CD34⁺ cells comprises using ananti-CD34 antibody conjugated to magnetic beads.
 66. The methodaccording to claim 61, further comprising expanding the CD34⁺ cells exvivo.
 67. The method according to claim 60, further comprising preparingmonocyte-derived pluripotent stem cells from the cell populationenriched for leukocytes.
 68. The method according to claim 60, furthercomprising inducing differentiation of a progenitor or stem cell. 69.The method according to claim 60, further comprising inducingtransdifferentiation of a differentiated cell.